As one of the most important staple crops, rice not only provides more than one fifth of daily calories for half of the world’s human population but is also a major source of mineral nutrients such as Mo, an essential micronutrient for almost all living organisms. In humans, Mo is required for the biosynthesis of molybdenum cofactor (Moco), which forms the active site of molybdenum-requiring enzymes (molybdoenzymes) including aldehyde oxidase, xanthine dehydrogenase, sulfite oxidase and amidoxime reducing component. These enzymes participate in crucial processes such as purine metabolism and sulfite detoxification and play a vital role in maintaining human healt. Deficiency of Moco biosynthesis in humans results in the decrease of molybdoenzyme activity, which leads to inheritable progressive neurological damage and even early childhood death. However, little is known about the genetic basis of the variation in Mo content in rice (Oryza sativa L.) grain. We mapped a quantitative trait locus (QTL) qGMo8 that controls Mo accumulation in rice grain by using a recombinant inbred line population and a backcross introgression line population. We identified a molybdate transporter, OsMOT1;1, as the causal gene for this QTL. OsMOT1;1 exhibits transport activity for molybdate, but not sulfate, when heterogeneously expressed in yeast cells. OsMOT1;1 is mainly expressed in roots and is involved in the uptake and translocation of molybdate under molybdate limited condition. Knock-down of OsMOT1;1 results in less Mo being translocated to shoots, lower Mo concentration in grains and higher sensitivity to Mo deficiency. We reveal that the natural variation of Mo concentration in rice grains is attributed to the variable expression of OsMOT1;1 due to sequence variation in its promoter. Identification of natural allelic variation in OsMOT1;1 may facilitate the development of rice varieties with Mo enriched grain for dietary needs and improve Mo nutrition of rice on Mo-deficient soils.